L2 practice problems

L2 practice problems - MCB121 W09 Practice problems for...

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Unformatted text preview: MCB121 W09 Practice problems for Lecture 2 1. A. You have purified a chromosome-associated protein in extracts prepared from cells in interphase. You are interested to know if this protein binds to one or more sequences or locations in the genome. Briefly outline the steps you would take to answer this question. B. It turns out that your protein is associated with repeated DNA sequences found at the ends of chromosomes. Where might these sequences be located in an interphase nucleus? C. You are able to purify enough protein to carry out mass spectrometry and determine some of its amino acid sequence. You use this peptide sequence to search a database of protein sequences and find that it is closely related to a family of histone deacetylases. Is this finding consistent with the cellular localization of this protein? 2. You are interested to know if your protein can deacetylate histones in vitro. You create a chromatin substrate using a 1:2 molar ratio of DNA to purified histones, including acetylated H4. You start with a fragment of DNA that is 2 kb long. How many total H4 substrates are likely to be present on your final product? Explain how you came up with your answer. KEY 1. A. You have purified a chromosome-associated protein in extracts prepared from cells in interphase. You are interested to know if this protein binds to one or more sequences or locations in the genome. Briefly outline the steps you would take to answer this question. You could use one of two methods. For both methods you will need to raise an antibody that binds specifically to the protein (this is typically done by injecting purified protein into rabbits and purifying the raised antibodies from its blood serum several weeks later). In method 1, cells fixed to a microscope slide can be treated with the antibody followed by addition of a secondary antibody that is conjugated to a fluorophore and binds to the Ig region of the first antibody. The protein of interest can microscopically visualized indirectly by exposing cells to UV light. For the second method you could use chromatin immunoprecipitation (ChIP). These steps include 1) crosslinking DNA and its bound protein, 2) lysing cells and shearing DNA to small (~500 bp fragments), 3) adding antibody of protein of interest followed by precipitation of the protein-DNA-antibody complex 4) removing crosslinks and analyze DNA products using pyrosequencing or by hybridization to microarrays. B. It turns out that your protein is associated with repeated DNA sequences found at the ends of chromosomes. Where might these sequences be located in an interphase nucleus? These sequences will likely be at the nuclear periphery since that is where telomeres are associated. C. You are able to purify enough protein to carry out mass spectrometry and determine some of its amino acid sequence. You use this peptide sequence to search a database of protein sequences and find that it is closely related to a family of histone deacetylases. Is this finding consistent with the cellular localization of this protein? Yes, heterochromatin is also found near the nuclear periphery. Heterochromatin is characterized by compact nucleosome spacing that and silenced gene expression. These properties are associated with deacetylated histones. 2. You are interested to know if your protein can deacetylate histones in vitro. You create a chromatin substrate using a 1:2 molar ratio of DNA to purified histones, including acetylated H4. You start with a fragment of DNA that is 2 kb long. How many total H4 substrates are likely to be present on your final product? Explain how you came up with your answer. Since the DNA required to bind one nucleosome is ~ 200 bp, it is likely that there will be 10 nucleosomes formed. Since every nucleosome has two H4 subunits there would be a total of 20 substrates that could be deacetylated. ...
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This note was uploaded on 04/06/2011 for the course MCB 121 taught by Professor Gasser during the Winter '09 term at UC Davis.

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